The thrust of this proposal is to attain novel and fundamental insights into the cellular uptake and intracellular trafficking of antisense and siRNA oligonucleotides and the implications for pharmacological effectiveness in cancer. We have synthesized oligonucleotide conjugates bearing high affinity ligands for Integrins, G-Protein Coupled Receptors, and other receptors. We can vary the ligand type, affinity, and valency. Using these reagents we will elucidate pathways of receptor- mediated cellular uptake and trafficking. We will also manipulate those pathways utilizing both chemical inhibitors and molecular tools such as activated or dominant-negative versions of proteins involved in trafficking. We will evaluate the impact of such manipulations on the pharmacologic effectiveness of antisense and siRNA via sensitive reporter assays. We will seek to identify the uptake and trafficking pathway(s) that provide the most effective delivery to the cytosol, and nucleus, and thus allow the greatest pharmacologic effect. These studies will be performed in three different contexts. Standard two-dimensional (2-D) culture of cancer cell lines will provide a setting where molecular manipulations and quantitative confocal fluorescence microscopy can be performed with relative ease. Three-dimensional (3-D) cancer cell cultures will be used as an important intermediate that may more closely reflect the situation in tumors, but that is still amenable to manipulation. Finally, via use of intravital microscopy and other tools, we will examine the uptake, distribution, and effect of antisense and siRNA oligonucleotides in xenograft tumors. The concerted use of 2-D, 3-D and in vivo systems will provide a rich stream of correlated information that will be of importance in the design of effective strategies for receptor-mediated delivery of therapeutic oligonucleotides in cancer.